1 Mycobacterium tuberculosis (Mtb) is a remarkably successful pathogen that causes high morbidity and mortality. There 2 is wide consensus that novel vaccines and vaccination strategies are essential to control this rampant disease. One such 3 strategy is inducing T-cell immunity to Mtb antigens that are presented by conserved, non-classical antigen presentation 4 molecules such as human HLA-E, CD1 and MR1. HLA-E is particularly interesting in the context of TB: (1) there are 2 5 coding variants that differ in only one amino acid outside the peptide binding groove; (2) HLA-E is relatively resistant to 6 HIV co-infection mediated downregulation; (3) HLA-E is enriched in Mtb phagosomes, facilitating its loading with Mtb 7 peptides; and (4) in mice, HLA-E (Qa-1b) restricted CD8+ T-cells are associated with protection. We have shown that 8 multiple Mtb epitopes can be presented by HLA-E and are potently recognized by Mtb specific CD8+ T-cells with unique 9 functional properties, including the ability to inhibit intracellular Mtb growth. The long-term goal of our work is to 10 understand the molecular interactions between peptide ligands, HLA-E and T-cell receptor (TCR) molecules in TB, and 11 to harness this knowledge into improved TB vaccination strategies. 12 The objective of our proposal is to define in detail the molecular interactions between HLA-E, peptide and TCR molecules 13 and to characterize HLA-E restricted T-cell responses against these peptides during infection and disease. To this end 14 we will define the exact molecular and structural determinants governing interactions between HLA-E, Mtb peptides 15 and TCR molecules (aim 1). In this aim we will investigate the molecular requirements governing peptide binding to both 16 HLA-E variant molecules and utilize that information to identify novel epitopes in the Mtb genome with improved HLA- 17 E binding and presentation properties. Secondly, available TCR sequences will be employed to generate TCR transduced 18 cell lines to assess critical peptide residues for TCR activation. The expected outcome of this aim is detailed insights in 19 the molecular interactions between HLA-E, peptide and TCR molecules that lead to productive TCR activation. 20 Secondly, we will define the functional and biological significance of Mtb peptide/HLA-E/TCR interactions in non-human 21 primates (NHP) and in human Mtb infection, vaccination and controlled human infection (CHI, with aerosolized BCG) 22 (aim 2). We will construct and use Mtb peptide/HLA-E tetramer pools to determine the frequency and functionality of 23 specific CD8+ T-cells in human samples from BCG vaccinees, latent or active Mtb infection, as well as BAL samples from 24 CHI, in which aerosol-BCG induced mucosal immune responses will be analysed for HLA-E restricted T-cell frequencies 25 and functionalities. To correlate HLA-E restricted T-cells with protective immunity, we will tetramer-profile samples 26 from banked NHP studies that included Mtb challenges with known outcome. The expected outcome of this aim is 27 insights in correlations of HLA-E restricted T-cells with infection, disease and vaccination in humans and NHP. 28 Together these results will impact and significantly deepen our understanding of the role of these non-classical T-cells 29 in Mtb infection, TB disease, vaccination and controlled human BCG infection, and pave the way towards harnessing 30 this knowledge for future TB vaccine design.